Catalytic RNAs and retrotransposons are key RNA elements that help shape genomes. Bacterial group II introns (gII-ins) are both self-splicing RNAs, which are the putative progenitors of spliceosomal introns, and mobile retroelements, and as such they occupy a pivotal role in early eukaryotic evolution. Yet, gII-ins are mysteriously absent from nuclear genomes, where we have shown them to silence gene expression. At a structural level, gII-in RNA combines with an intron-encoded protein to form a ribonucleoprotein (RNP) that is active in both splicing and mobility. The overall goal of this application is to understand the structure and function of these bacterial mobile self-splicing retroelements, while relating them to their eukaryotic spliceosomal and retroelement counterparts. This goal will be achieved by combining interdisciplinary biochemical, biophysical, cellular, computational, genetic, structural and systems approaches. The work will be performed in an array of model organisms, including the bacterium Lactococcus lactis and the yeast Saccharomyces cerevisiae. Considerable progress over the past funding period, including structural analysis of the gII-in RNP, and functional studies in bacteria and yeast, is the springboard for three proposed specific aims: Aim 1: To capture structure transitions of the gII-in RNP in splicing and gene targeting. We will use native RNPs purified from L. lactis to develop a series of structural and kinetic snapshots of the gII-in RNP at different stages. These include the loose RNP precursor; the compact, spliced free intron RNP; and the free intron RNP attacking its DNA target for retromobility. Aim 2: To define silencing of gII-in-containing genes in eukaryotes. We will dissect the process of silencing of gene expression imposed by the gII-in, which is thought to have played a role in the gII- in/spliceosomal intron transition, by defining host factors that result in RNA mislocalization. Thus, by combining yeast genetics, RNA biochemistry, cell and systems biology, we will establish a mechanistic appreciation of why gII-ins were expunged from eukaryotic nuclei. Aim 3: To understand host-retrotransposon relationships across kingdoms. We will define the dynamic relationship, both positive and negative, between the parasitic gII-in and the bacterial host, L. lactis, using biochemical, genetic and systems approaches. These studies will be integrated with the NIH- funded Center for Systems Biology of Retrotransposition, which focuses on mammalian retrotransposons. The impact of our trans-kingdom approach is an enhanced understanding of the structure and function of self-splicing elements that have been exploited biotechnologically to edit genes, and which resemble retrotransposons that sculpt diverse genomes in health and disease.